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Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii.

ABSTRACT: 1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhibitory agent was purified to homogeneity by anion-exchange, cation-exchange, gel-filtration and reverse-phase chromatography to yield a single band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments showed tridegin to be a peptide of 66 amino acids. The sequence is unique with little similarity to other leech-derived proteins. 4. Inhibition of plasma factor XIIIa activity was confirmed by four independent methods: tridegin increased the solubility of fibrin clots in urea, inhibited ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5'-(biotinamido)pentylamine into casein and prevented gamma-dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration of factor XIIIa used in the assay and in fact depends on its concentration. This is the most potent inhibitor of factor XIIIa yet described. 6. Tridegin also inhibits platelet factor XIIIa (factor XIIIAa) with a similar potency to that of the plasma enzyme. 7. Tridegin also inhibits tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for transglutaminases, since it has no effect on the coagulation times of human plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or cleave the isopeptide substrate, L-gamma-glutamyl-4-nitroanilide.

SUBMITTER: Finney S

PROVIDER: S-EPMC1218495 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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Project description:Leeches are found in terrestrial, aquatic, and marine habitats on all continents. Sanguivorous leeches have been used in medicine for millennia. Modern scientific uses include studies of neurons, anticoagulants, and gut microbial symbioses. Hirudo verbana, the European medicinal leech, maintains a gut community dominated by two bacterial symbionts, Aeromonas veronii and Mucinivorans hirudinis, which sometimes account for as much as 97% of the total crop microbiota. The highly simplified gut anatomy and microbiome of H. verbana make it an excellent model organism for studying gut microbial dynamics. The North American medicinal leech, Macrobdella decora, is a hirudinid leech native to Canada and the northern United States. In this study, we show that M. decora symbiont communities are very similar to those in H. verbana. We performed an extensive study using field-caught M. decora and purchased H. verbana from two suppliers. Deep sequencing of the V4 region of the 16S rRNA gene allowed us to determine that the core microbiome of M. decora consists of Bacteroides, Aeromonas, Proteocatella, and Butyricicoccus. The analysis revealed that the compositions of the gut microbiomes of the two leech species were significantly different at all taxonomic levels. The R2 value was highest at the genus and amplicon sequence variant (ASV) levels and much lower at the phylum, class, and order levels. The gut and bladder microbial communities were distinct. We propose that M. decora is an alternative to H. verbana for studies of wild-caught animals and provide evidence for the conservation of digestive-tract and bladder symbionts in annelid models.IMPORTANCE Building evidence implicates the gut microbiome in critical animal functions such as regulating digestion, nutrition, immune regulation, and development. Simplified, phylogenetically diverse models for hypothesis testing are necessary because of the difficulty of assigning causative relationships in complex gut microbiomes. Previous research used Hirudo verbana as a tractable animal model of digestive-tract symbioses. Our data show that Macrobdella decora may work just as well without the drawback of being an endangered organism and with the added advantage of easy access to field-caught specimens. The similarity of the microbial community structures of species from two different continents reveals the highly conserved nature of the microbial symbionts in sanguivorous leeches.

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Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii.

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